PEP is a high energy intermediate of glycolysis, gluconeogenesis, and the C4 plant cycle for concentrating CO2. The high energy phosphate of PEP contains sufficient for substrate level phosphorylation of ADP to form ATP and pyruvate, as shown below:

Phosphoenolpyruvate + ADP + H⁺ <=> Pyruvate + ATP (catalyzed by Pyruvate Kinase)

PEP functions in the PEP-dependent phosphotransferase system (PTS) of E. coli , which transports and phosphorylates sugars. PEP is used in synthesis of N-acetylneuraminic acid 9-phosphate (Figure 16.14)

Enzymes that act on PEP include PEPCK, enolase, pyruvate kinase, pyruvate-phosphate dikinase, PEP Carboxylase, and 5-enoylpyruvaylshikimate-3-phosphate synthase (EPSP synthase)